Microorganisms
Chatemouim globosum is a local fungal strain isolated from Egyptian soil. It was identified in Plant Pathology Department of National Research Centre, Giza, Egypt, according to Barnett and Hunter (1972) and Nelson et al. (1983). The strain was kept on a potato dextrose agar medium and stored at 4 °C.
Three fungal pathogens F. solani, M. phaseolina, and R. solani were selected from preliminary test of isolated from legume seeds The fungi in pure culture were identified after pathogenicity test according to the keys given by Barnett and Hunter (1972) and Nelson et al. (1983).
Common legume seed samples were collected in polythene envelopes from different market from Egypt and taken to the laboratory for isolation of fungal pathogens.
Substrates
Different agricultural residues (banana peel, pea peel sawdust, peanuts, pomegranate peel (Pp), potato peel, wheat straw, sugar cane bagasse, soy bean) were used as sole carbon source and tested for xylanase production. All wastes were washed, dried at 70 °C in oven, and cut into small pieces (1 cm × 1 cm) before used.
Fermentation condition
The ability of the microorganism for xylanase production was examined in medium containing (g/l) NaNO3 2.0, K2HPO4 0.5, KCl 0.5, MgSO4 0.7, H2O 0.5, and different agricultural wastes (20.0). Erlenmeyer flasks 250 ml each containing 50 ml fermentation medium were inoculated with two disks 6 mm in diameter of the selected microorganism and incubated at 28 ± 30 °C for 7 days in incubator shaker at 200 rpm.
Xylanase assay
Determination of enzyme activity was carried out according to the method of Monreal and Reese (1969).
One milliliter of 1% birch wood xylan (Sigma) in acetate buffer (pH 4.6) in test tubes was added to 1 ml of the culture filtrate and mixed by shaking. The mixture was incubated in a water bath at 50 °C for 30 min and then cooled and centrifuged before assaying. The amount of reducing sugar was determined with 1 ml of 3,5-dinitrosalicylic acid (DNS). One unit of enzyme activity was taken of the catalyst converts one μmol of substrate in 1 min under specific condition.
Effect of different agricultural residue
In order to induce xylanase synthesis from microbial sources from cheap and most economic carbon source, 20 g of different agricultural residues (banana, pea peal, sawdust, peanuts, pomegranate peel, potato peel wheat straw, sugar cane baggase, and soy bean) in comparison with control was added to fermentation medium.
Optimization of culture conditions
Optimization conditions were carried out depends on addition of different (Pp) concentrations ranging from 5 to 120 g/l. Different inoculum size 2 disks (2, 4, 6, and 8 mm) in diameter, different incubation period (5, 7, and 10) days, and addition of different salts (sodium bicarbonate, calcium carbonate, potassium sorbate, calcium chloride) were obtained from Sigma Aldrich Spruce Street, St. Louis, USA.
Characterization of crude xylanase activity
Optimization of crude xylanase activity was carried out using crude filtrate of C. globosum. Effect of different reaction time was carried out in the reaction mixture ranging from 10 to 60 min, followed by changing pH values from 3.6 to 7.0. The temperature of the reaction was analyzed by incubated reaction for 10–60 °C.
Thermal stability
Thermal stability was analyzed by incubating the crude filtrate at different temperature with different time intervals, and then, residual activity was measured.
Collection of the seed samples
Twenty seed samples are from 5 leguminous crop, namely, Pisum sativm (pea), Vicia faba (faba bean), Cicer arietinum (chickpea), Phaseolus vulgaris (white bean), and Lens culinaris (lentil).
Seed Borne fungi analysis
Detection of seed borne fungi from selected legume seeds was done by agar plate method as recommended by ISTA (1976).
Frequency of occurrence of isolated fungi
Frequency of fungal isolates was calculated according to Al-Abdalall (2008).
$$ \%\mathrm{Frequency}=\mathrm{total}\ \mathrm{number}\ \mathrm{of}\ \mathrm{fungal}\ \mathrm{isolates}/\mathrm{crop}/\mathrm{number}\ \mathrm{of}\ \mathrm{fungal}\ \mathrm{isolates}\times 100. $$
Evaluation of the efficacies of crude xylanase produced against different fungal isolates
Agar well diffusion method crude xylanase from C. globosum was screened for antifungal activity using sterile cork borer of size 6.0 mm in diameter according to Bobbarala et al. (2009). Five hundred microliters of crude xylanase solution homogenized added to 0.02 ml of inoculums filled in deep blocks. Incubation period of 48–72 h at 25 °C was maintained for antifungal activity. Radial inhibition was calculated when growth of mycelia in the control plate reached the edge of the petri dish. The toxicity of the extracts to growth of fungi was calculated by using the formula percentage (%) inhibition = dc − dt/dc × 100, where dc = average increase in mycelial growth in control and dt = average increase in mycelial growth in treatment (Singh and Tripathi 1999)
Greenhouse experiment
Three fungal pathogens F. solani, M. phaseolina, and R. solani were selected from preliminary test of isolated from legume seeds which showed the highly percentage of occurrence and evaluate the biocide prepare from C. globosum grown on Pp waste under greenhouse conditions for bean seed P. vulgaris. Sandy clay soil was transferred in pots. Inoculum from each cultures was colonized separately and infested at the rate of 3 g/100 g soil. The disinfected bean seeds were coated with a biocide at the rate of 4 ml/kg seeds. Seed dressing was carried out by applying the biocide to the gum moistened seeds in polyethylene bags and shaking well to ensure even distribution of the added materials then left to air dried. Five replicates/treatments, pathogen free seeds were surface sterilized and planted (5 seeds/pot) in inoculated and non-inoculated soils. All pots maintained in greenhouse under natural condition, for 15 days after sowing. Disease ratios were determined by recording the number of non-emerged seeds (pre-emergence damping-off) while post-emergence damping off was recording from 30 to 45 days after sowing. The equations described by Khalifa (1987) were as follows:
$$ \mathrm{Pre}-\mathrm{emergence}\ \left(\%\right)\ \mathrm{damping}\ \mathrm{off}=\mathrm{no}.\mathrm{of}\ \mathrm{non}-\mathrm{emerged}\ \mathrm{seeds}/\mathrm{no}.\mathrm{of}\ \mathrm{sown}\ \mathrm{seeds}\times 100 $$
$$ \mathrm{Post}-\mathrm{emergence}\ \left(\%\right)\ \mathrm{damping}\ \mathrm{off}=\mathrm{no}.\mathrm{of}\ \mathrm{killed}\ \mathrm{seedling}/\mathrm{no}.\mathrm{of}\ \mathrm{sown}\ \mathrm{seeds}\times 100 $$
Statistical analysis
Tukey test for multiple comparisons among means was utilized (Neler et al. 1985).